An HPLC assay with fluorometric detection has been developed which is sensitive enough to simultaneously measure endogenous tryptophan, serotonin (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) inside of synaptosomes as well as that which has been released into the incubation medium. Using this assay, we have observed that tryptophan is rapidly taken up by synaptosomes and turned over to 5-HIAA without a concurrent release of 5-HT. Exogenous 5-HT is also rapidly taken up and, within 20-30 min, 80% of the 5-HT is deaminated. Veratridine releases both tryptophan and 5-HT from synaptosomes. Changes in the disposition of exogenous tryptophan or 5-HT can be completely accounted for by uptake or by stoichiometric changes in metabolites. This assay method should be valuable in the study of 5-HT pools and in the determination of from which pool 5-HT release occurs.